large ring polymers align ftsz polymers for normal septum formation

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Large ring polymers align FtsZ polymers fornormal septum formation

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Muhammet E Gundogdu1,6,Yoshikazu Kawai1,2,6, Nada Pavlendova1,3,6,Naotake Ogasawara2, Jeff Errington1,Dirk-Jan Scheffers4,5 andLeendert W Hamoen1,*1Centre for Bacterial Cell Biology, Institute for Cell and MolecularBiosciences, Newcastle University, Newcastle, UK, 2Nara Instituteof Science and Technology, Graduate School of Information ScienceFunctional Genomics, Ikoma, Japan, 3Institute of Molecular Biology,Slovak Academy of Sciences, Bratislava, Slovak Republic, 4BacterialMembrane Proteomics Laboratory, Instituto de Tecnologia Quımica eBiologica, Universidade Nova de Lisboa, Oeiras, Portugal, and5Department of Molecular Microbiology, Groningen BiomolecularSciences and Biotechnology Institute, University of Groningen, NNHaren, The Netherlands

Cytokinesis in bacteria is initiated by polymerization of

the tubulin homologue FtsZ into a circular structure at

midcell, the Z-ring. This structure functions as a scaffold

for all other cell division proteins. Several proteins support

assembly of the Z-ring, and one such protein, SepF, is

required for normal cell division in Gram-positive bacteria

and cyanobacteria. Mutation of sepF results in deformed

division septa. It is unclear how SepF contributes to

the synthesis of normal septa. We have studied SepF by

electron microscopy (EM) and found that the protein

assembles into very large (B50 nm diameter) rings.

These rings were able to bundle FtsZ protofilaments into

strikingly long and regular tubular structures reminiscent

of eukaryotic microtubules. SepF mutants that disturb

interaction with FtsZ or that impair ring formation are

no longer able to align FtsZ filaments in vitro, and fail to

support normal cell division in vivo. We propose that SepF

rings are required for the regular arrangement of FtsZ

filaments. Absence of this ordered state could explain

the grossly distorted septal morphologies seen in sepF

mutants.

The EMBO Journal (2011) 30, 617–626. doi:10.1038/

emboj.2010.345; Published online 11 January 2011

Subject Categories: microbiology & pathogens

Keywords: cell division; FtsA; FtsZ; SepF; Z-ring

Introduction

The earliest known event in bacterial cell division is the

assembly of the tubulin-like protein FtsZ into a circular

structure at midcell (Adams and Errington, 2009). This so-

called Z-ring recruits the other proteins needed for synthesis

of the division septum. How the Z-ring is structured in the

cell is not really known. Electron microscopy (EM) studies

have shown that, depending on the reaction conditions,

purified FtsZ can polymerize into long bundles, and struc-

tures like sheets, mini-rings, and helices (Bramhill and

Thompson, 1994; Mukherjee and Lutkenhaus, 1999; Lu

et al, 2000; Popp et al, 2009). Assembly of FtsZ into a stable

Z-ring at the site of cell division involves several other

proteins. One key player is FtsA, which binds FtsZ and

links the Z-ring to the membrane via an amphipathic

a-helical domain (Jensen et al, 2005; Pichoff and Lutkenhaus,

2005). Another conserved protein, ZapA, forms a link be-

tween FtsZ protofilaments and stimulates polymerization

(Gueiros-Filho and Losick, 2002; Small et al, 2007). In rod-

shaped bacteria the Min proteins exercise a regulatory role,

and inhibit polymerization of FtsZ close to cell poles (Hu

et al, 1999; Scheffers, 2008). Gram-positive bacteria use

another FtsZ regulator, the integral membrane protein EzrA

(Levin et al, 1999). Deletion of this protein leads to extra

Z-rings, and it is therefore considered a negative regulator. In

this study we investigated SepF (YlmF), a Z-ring-associated

protein that is highly conserved in Gram-positive bacteria and

cyanobacteria (Miyagishima et al, 2005; Hamoen et al, 2006;

Ishikawa et al, 2006).

The first indications that SepF has a role in cell division

came from studies with Streptococcus pneumoniae and the

cyanobacterium Synechococcus elongatus, which showed that

mutations in sepF lead to severe cell division defects (Fadda

et al, 2003; Miyagishima et al, 2005). Further studies with

Bacillus subtilis revealed that SepF localizes to the division

site. This localization depended on the presence of FtsZ, and

yeast two-hybrid experiments showed a direct interaction

between both proteins (Hamoen et al, 2006; Ishikawa et al,

2006). Deletion of sepF results in grossly deformed division

septa in B. subtilis. Interestingly, deformed septa are not

observed with other cell division mutants. SepF has an

apparent functional overlap with FtsA. FtsA is not essential

in B. subtilis, although B. subtilis ftsA mutants grow slower

and form filamentous cells (Beall and Lutkenhaus, 1992;

Jensen et al, 2005). This phenotype can be restored by

overexpression of sepF. A double disruption of sepF and

ftsA completely eliminates Z-ring formation and is lethal.

This functional overlap with FtsA led Ishikawa et al (2006) to

conclude that, like FtsA, SepF is involved in the early stages

of Z-ring assembly. However, these results seem to contradictReceived: 22 July 2010; accepted: 29 November 2010; publishedonline: 11 January 2011

*Corresponding author. Institute for Cell and Molecular Biosciences,Centre for Bacterial Cell Biology, Newcastle University, RichardsonRoad, Framlington Place, Newcastle NE2 4AX, UK.Tel.: þ 44 191 208 3240; Fax: þ 44 191 208 3205;E-mail: [email protected] authors contributed equally to this work

The EMBO Journal (2011) 30, 617–626 | & 2011 European Molecular Biology Organization | Some Rights Reserved 0261-4189/11

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our earlier data on SepF. If SepF stimulates polymerization

of FtsZ one may assume that a sepF mutant becomes sensitive

for reduced FtsZ levels, but this is not the case (Hamoen

et al, 2006). Furthermore, if SepF supports Z-ring formation

it is unlikely that deletion of a negative regulator of FtsZ

polymerization would cause problems, and yet introduction

of a sepF mutation in an ezrA deletion background proved

to be lethal.

It is unclear at what stage of the division process SepF is

active, and to gain more information on this we isolated

dominant negative sepF mutants. To test whether the muta-

tions affected the interaction with FtsZ, we purified SepF.

This resulted in a rather striking observation. It turned out

that SepF assembles into very large rings that can bundle FtsZ

protofilaments into long and regular tubular structures.

The dominant negative sepF mutants were unable to form

these tubules. A mutation that blocked ring formation was

also unable to align FtsZ protofilaments. The results support

a new model in which SepF forms regular ring polymers

that organize FtsZ protofilaments into higher order structures

required for a smooth invagination of the Gram-positive

septal wall. Thus, SepF is more than a simple positive

regulator of Z-ring formation, which might explain why its

absence is synthetic lethal in an ftsA mutant as well as in an

ezrA mutant.

Results

SepF polymerizes into very large rings

To investigate in detail how SepF influences the polymeriza-

tion of FtsZ, we decided to purify both proteins for biochem-

ical analysis. SepF was purified using maltose-binding

protein (MBP) as affinity tag. The MBP moiety was cleaved

from SepF and removed by ion-exchange chromatography.

When subjected to size exclusion chromatography, SepF

eluted in the void volume, suggesting that the protein formed

aggregates. To confirm this, we examined the protein sample

by EM, yet the EM images revealed a very surprising result.

It proved that SepF polymerizes into very large regular

ring structures with an average diameter of about 50 nm

(Figure 1). These rings are so wide that, theoretically, they

can encompass two whole ribosomes. The formation of these

rings seemed a robust process as they were readily formed

at high salt concentrations (500 mM), and in the presence

of other proteins (e.g., BSA). Although the buffer used in

Figure 1 contains Mg2þ (see below), this is not required for

ring formation. Rings were still visible at SepF concentrations

as low as 0.1 mM, but their number was greatly reduced, and

they were difficult to find on the EM grid. It was therefore not

possible to determine the critical concentration at which rings

were formed. The ring structures display a limited variability

in ring diameter (Figure 1). This suggests that SepF filaments,

presumptive precursors of the closed rings, are rather inflex-

ible and always close on themselves.

SepF is an abundant protein

The striking SepF rings begged the question what effect they

would have on FtsZ polymerization. To analyze this under

physiological conditions, we first determined the ratio of

both proteins in the cell. Antibodies against purified SepF

were raised and used to perform a quantitative western blot

analysis. For exponentially growing cells, we estimated that

the cellular concentrations of SepF and FtsZ were B6 and

8mM, respectively (Supplementary Figure S1). This roughly

correspond to 8000 molecules of SepF per cell, and 11000

molecules for FtsZ, assuming a cell volume of 2.6 mm3

(Henriques et al, 1998). FtsZ is an abundant protein in the

cell (Wang and Lutkenhaus, 1993), and the fact that the

amount of SepF is almost as high as that of FtsZ suggests

that the SepF rings have a structural role rather than a

regulatory role in Z-ring formation.

SepF-ring formation requires physiological reaction

conditions

FtsZ polymerization can be followed by pelleting and

light scattering assays (Mukherjee and Lutkenhaus, 1998;

Scheffers, 2008). When purified SepF was included in an

FtsZ polymerization experiment a small increase in the

DC

A B

0

5

10

15

20

25

40 45 50 55 60 65

%

Diameter (nm)

Figure 1 SepF forms large ring structures. (A) EM images of negatively stained SepF rings (6mM SepF in buffer: 50mM Tris–HCl pH 7.4, 300 mMKCl, 10mM MgCl2). (B) SepF rings at higher magnification. (C) Histogram showing the distribution of SepF ring diameters (213 rings counted).(D) SepF in polymerization buffer of pH 6.5 (50 mM MES pH 6.5, 50mM KCl, 10mM MgCl2). Scale bars: 100nm (A, D), and 50nm (B).

SepF rings align FtsZ protofilamentsME Gundogdu et al

The EMBO Journal VOL 30 | NO 3 | 2011 &2011 European Molecular Biology Organization618

amount of pelleted FtsZ was observed, suggesting that SepF

stimulates polymerization of FtsZ (Figure 2A, lanes 1 and 2).

However, we noticed a problem with the solubility of SepF.

FtsZ polymerization assays are generally performed at a

relatively low pH of around pH 6.5, as this stimulates the

lateral association of FtsZ protofilaments (Mukherjee and

Lutkenhaus, 1999). It emerged that the low pH resulted in

precipitation of SepF, and EM images of SepF in a buffer of pH

6.5 showed that protein rings were no longer present

(Figure 1D). Precipitation of SepF was reduced by increasing

the salt concentration (300 mM KCl) and using a buffer with a

more physiological pH of 7.4 (Booth, 1985; Breeuwer et al,

1996) (Supplementary Figure S2B). This effect did not de-

pend on the type of buffer used, and the same result was

obtained with a Tris, HEPES or MES buffer (data not shown).

At pH 7.4 and 300 mM salt, the total amount of pelleted FtsZ

was substantially less, but the stimulatory effect of SepF on

pelleting was again detectable (Figure 2A, lanes 3 and 4). In

light scattering assays using standard polymerization buffer

of pH 6.5, B. subtilis FtsZ gave a classic response upon

the addition of GTP, with a strong increase in light scatter

signal that decreased over time (Supplementary Figure S3)

(Mukherjee and Lutkenhaus, 1999). The light scattering

signal was strongly reduced by a shift to pH 7.4, and almost

eliminated by increased salt concentrations (Supplementary

Figure S3), indicating that lateral interactions between

FtsZ protofilaments are greatly diminished under these

conditions. The addition of SepF did result in a shift in

light scattering signal (Figure 2B) but not the gradual rise

that is normally associated with bundling of FtsZ protofila-

ments. SepF had no measurable effect on the GTPase activity

of FtsZ (Figure 2C).

SepF bundles FtsZ into tubules

We returned to EM to investigate whether the SepF rings

had any effect on FtsZ filaments. In a buffer of pH 7.4 with

300 mM KCl, FtsZ forms clear protofilaments when GTP is

present (Figure 3A). The addition of SepF had a remarkable

result and long regular tubular structures became visible

(Figure 3B). The average diameter of these structures was

about 48 nm (Figure 3C), close to the diameter of SepF rings.

Tubule formation was not influenced by the order in which

the proteins or GTP were added, but depended on the

concentration of FtsZ. When half the amount of FtsZ was

used (5 mM) less tubules were detected. Increasing the SepF

concentration did not compensate for this. At half the SepF

concentration (3 mM) tubules were still visible, but at 1mM

SepF and 10mM FtsZ no tubules could be identified on the EM

grids. When GDP instead of GTP was used no tubules were

formed. We never observed tubules in polymerization buffer

of pH 6.5, which may explain why they were not observed in

a previous study (Singh et al, 2008). We used 10 mM Mg2þ in

the polymerization buffers, which is commonly used for FtsZ

polymerization studies, but 1 mM Mg2þ was sufficient to see

tubules. Without Mg2þ no tubules were formed. At higher

magnifications the tubules appear to consist of straight long-

itudinal filaments, most likely FtsZ polymers (Figure 3D,

narrow arrows), with evident transverse bands that presum-

ably represent SepF rings (Figure 3D, wide arrows). From the

EM images it seems that the SepF rings form the core of the

FtsZ–SepF tubules, but attempts to resolve this question

using thin section EM or Cryo-EM were unsuccessful. The

length of the tubules increased with time and tubules could

grow up to micrometres in length (Figure 3E; Supplementary

Figure S4). Interestingly, during the first 5 min of the reaction

filaments emanating from the ends of the short tubules

tended to be straight (Figure 4A and B), whereas after

A

B

24

16

8

0

% P

elle

ted

Fts

ZpH 6.5

pH 7.4

FtsZ

FtsZ + SepF

C

90

1 2 3 4

60

30

00 3015

0 3015

Time (min)

a

b

c

Ligh

t sca

tter

(a.u

.)

900

600

300

0

Time (min)

FtsZ

FtsZ + SepF

Pi r

elea

se (

µM)

Figure 2 Biochemical analyses of the effect of SepF on FtsZ poly-merization. (A) Results from three independent FtsZ sedimentationexperiments. The increase in pelleting after addition of GTP(lanes 1 and 3) or GTP and SepF (lanes 2 and 4) are shown(see Supplementary Figure S2A for SDS–PAA gel). Lanes 1 and 2are samples prepared in pH 6.5 polymerization buffer (50 mM MESpH 6.5, 50 mM KCl, 10 mM MgCl2), and lanes 3 and 4 are samplesprepared in pH 7.4 buffer (50 mM Tris–HCl pH 7.4, 300 mM KCl,10 mM MgCl2). (B) Light scattering analyses of FtsZ polymerizationin pH 7.4 buffer. FtsZ (a), SepF (b), and GTP (c) were added insubsequent steps. (C) GTP hydrolysis during FtsZ polymerization inpH 7.4 buffer (average of four independent experiments). In allexperiments 1 mM GTP, 10mM FtsZ, and 6 mM SepF were used.


&2011 European Molecular Biology Organization The EMBO Journal VOL 30 | NO 3 | 2011 619

10 min, some tubules had splayed tips with highly curved

filaments (Figure 4C–E). In the later samples (20 min), tub-

ules with different kind of bifurcations, including branches

and rings, were evident (Figures 3E and 4F–I).

Screen for SepF mutations that inhibit cell division

Although the formation of FtsZ–SepF tubules appeared a

reproducible and robust process in vitro, it was important

to test its physiological relevance. We investigated whether it

was possible to isolate dominant negative mutations in SepF

that would affect the interaction of SepF with FtsZ and so

disrupt tubule formation. The screen took advantage of the

fact that a combination of sepF and ftsA null mutations is

lethal (Ishikawa et al, 2006). Randomly mutagenized alleles

of sepF were expressed from a xylose inducible Pxyl-promoter

in cells carrying a wild-type sepF gene and a deletion of ftsA.

After testing several thousands of transformants two clones

were identified that were unable to grow when the sepF

mutant allele was overexpressed by the addition of xylose

to the growth medium (Figure 5A). Sequencing of the mu-

tated sepF alleles revealed two different mutations affecting

conserved residues: A98V and F124S (Figure 6). When

these SepF mutants were (over)expressed in a strain devoid

of wild-type SepF, cells remained elongated, and there was

no indication that the mutant proteins could compensate

for the absence of wild-type protein. To test whether the

dominant negative sepF mutants affected Z-ring formation,

we examined the localization of a gfp–ftsZ fusion. As shown

in Figure 5B (upper panel), the overexpression of wild-type

SepF in an ftsA mutant background had no effect on Z-ring

formation. However, when the mutants A98V or F124S

were induced (Figure 5B, lower panels), Z-ring formation

was abolished, indicating that the mutant proteins inhibit cell

division by interfering with Z-ring formation.

Dominant negative SepF mutants do not form tubules

The inability of the SepF mutants to support growth could be

a consequence of mutated FtsZ-binding sites or possibly a

failure to form ring structures. To test this, the mutant SepF

proteins were purified and first analyzed by size exclusion

chromatography. Wild-type SepF showed a large peak in the

void volume (42 MDa), probably corresponding to SepF

rings (Figure 7A). The F124S mutant showed a comparable

elution profile to the wild-type protein. However, the A98V

mutant was almost absent from the void volume, suggesting

that this mutant affects ring formation. This protein showed

an increased elution at around 14 ml between the peaks for

the reference proteins thyroglobulin (669 kDa) and aldolase

(158 kDa). Thus, the A98V mutation still gives oligomeric

structures and does not reduce the protein to its monomeric

state of 17 kDa. EM analysis showed normal ring formation

for the F124S mutant (Figure 7B). In case of the A98V mutant

0

5

10

15

20

25

37.5 42.5 47.5 52.5 57.5

%

Width (nm)

BA C

D

E

Figure 3 EM images of negatively stained FtsZ tubules formed by SepF rings. (A) FtsZ protofilaments in polymerization buffer pH 7.4 after theaddition of 1 mM GTP and (B) under the same conditions in the presence of SepF. Concentrations used for FtsZ and SepF were 10 and 6 mM,respectively. (C) Histogram showing the distribution of FtsZ–SepF tubule widths (148 measurements). (D) Detailed picture of two FtsZ–SepFtubules. In the upper tubule SepF rings are indicated by arrowheads, and in the lower tubule longitudinal FtsZ filaments are indicated byarrowheads. (E) FtsZ–SepF tubules can grow up to a few micrometres in length. Scale bars: 100 nm (A, B), 50 nm (D), and 500 nm (E).



it was difficult to find rings on the EM grids, indicating that

this mutant protein is indeed disturbed in ring formation.

We then examined the ability of the mutant proteins to

support the formation of FtsZ tubules. Importantly, neither of

the mutant proteins formed any detectable tubular structures

under conditions in which wild-type SepF supported abun-

dant tubulation (Supplementary Figure S5). As the F124S

protein still forms rings, it is possible that this mutant is

unable to interact with FtsZ. To test this, we performed an

FtsZ–SepF co-elution experiment using MBP–SepF fusions.

MBP–SepF, when bound to amylose resin, is capable of

selectively binding FtsZ from an extract of Escherichia coli

cells expressing B. subtilis FtsZ (Figures 7C and 8A). MBP–

SepF mutant fusions, bound to amylose resin, retained sig-

nificantly less FtsZ compared with wild-type protein. In fact,

the amount of FtsZ that interacted with these mutant proteins

was only detectable by western blot analysis. Thus, both

A98V and F124S mutations impair the interaction with FtsZ

protein. GFP fusions with these SepF mutants showed diffuse

fluorescence in cells, in agreement with a loss of FtsZ-binding

activity (data not shown).

The F124S mutant largely retained the ability to form rings,

and it is likely that its phenotype is manifested mainly

through impaired interaction with FtsZ. Possibly, this protein

interferes with wild-type SepF by creating hybrid rings that

are less efficient in bundling FtsZ protofilaments. The A98V

mutant could have a similar effect. We have tried to visualize

hybrid rings by employing SepF fusion proteins, such as an

N-terminal MBP–SepF fusion or a C-terminal SepF–Intein

fusion, but both fusion proteins were unable to form rings

by themselves. This could also explain why GFP fusions of

SepF are not active in vivo. Indeed, when we examined

purified SepF–GFP by EM no protein rings were detected

(data not shown).

Breaking the ring abolishes tubule formation

The above results suggest that alignment of FtsZ protofila-

ments is a key activity of SepF. However, it is still unclear

whether the formation of rings is important for this activity.

The inability of a SepF–GFP fusion to form rings indicates

that the C-terminus is probably important for ring formation.

Indeed, removal of the putative a-helical domain at the

C-terminus (mutant D134) abolishes formation of SepF rings

and of SepF–FtsZ tubules, even though the truncated

SepF can still bind FtsZ (Figure 8A, data not shown). The

first residue in this 15 amino acid C-terminal domain is a

conserved glycine (Figure 6). Mutation of this glycine to

asparagine (G135N) had no consequences for SepF–FtsZ

interaction as the mutant protein still bound FtsZ in vitro

(Figure 8A), and localized to the Z-ring in vivo (Figure 8B).

When we analyzed purified G135N mutant protein by EM no

protein rings were detectable, instead the protein appeared to

assemble into long filamentous structures (Figure 8C, left

panel). Importantly, mixing the mutant protein with purified

FtsZ did not result in tubular structures (Figure 8C, right

panel). These data suggest that the C-terminus of SepF

is required for the formation of rings, and that SepF rings

are required for the alignment of FtsZ polymers. To test

whether the sepF-ring mutant was active in vivo, we replaced

wild-type sepF with sepF–G135N in a B. subtilis strain

that contains ftsA under control of the IPTG-inducible Pspac

promoter. When the resulting strain was grown on plates

G H I

A B C

D E F

Figure 4 Compilation of EM images showing FtsZ–SepF tubule ends at different time points in the reaction. (A, B) Tubule ends after 5 minshowing relative straight filaments. (C–E) Curved ends are observed after about 10 min. (F, G) Some tubule ends show bifurcations after 20 minincubation. (H, I) Detail of branching FtsZ–SepF tubules. Scale bar: 100 nm.



without IPTG, no colonies appeared (Figure 8D), despite the

fact that expression levels of SepF–G135N were normal

(Figure 8E). The mutant, when overexpressed in an ftsA

deletion strain, showed no dominant negative effect like the

SepF mutants A98V and F124S. The finding that SepF–G135N

cannot support cell growth in the absence of FtsA indi-

cates that SepF-ring formation is critical for the function of

SepF, and lends further support to the conclusion that the

Figure 6 Amino acid sequence alignment of SepF proteins, from B. subtilis (Bsub), Listeria monocytogenes (Lmon), Staphylococcus aureus(Saur), and Streptomyces coelicolor (Scoe), using ClustalW. Secondary structure prediction (using PSIPRED) for B. subtilis SepF is shown abovethe sequences. The position of a-helices and b-sheets are indicated by rods and arrows, respectively. Conserved amino acids are marked in grey,and the mutated amino acids are indicated above the sequence (A98V, F124S, and G135N). In mutant D134, the protein is truncated at aminoacid 134.

A

wt

F124S

A98V

B

wt

A98V F124S

Wild type �ftsA

+ Xylose + Xylose

Figure 5 Isolation of SepF mutations that inhibit cell division.(A) The isolated mutations A98V and F124S were lethal whenexpressed (þ xylose) in a DftsA background. Strains were platedin the presence or absence of 2% xylose. The background genotypesare indicated above the plates. (B) Localization of GFP–FtsZ afterexpression of the SepF mutants (1% xylose) in a DftsA background(deletion of ftsA reduces cell division and leads to elongated cells).Scale bar: 5 mm.

A98V F124S

B

C wt– + – + – +

F124SA98V

A10

8

6

4

2

0 15 18 21 2412963

V (ml)

AU

wtF124SA98V

AT

Figure 7 Effects of mutations on ring formation and FtsZ binding.(A) Size exclusion chromatography of the different SepF mutantsusing an analytical Superose 6 column. The absorbance at 280 nm(AU) is plotted as a function of the elution volume. The peaks of tworeference proteins thyroglobulin (T, 669 kDa) and aldolase (A,158 kDa) are indicated by arrows. SepF has a calculated size of17 kDa. (B) EM images of negatively stained SepF mutants. Scalebar is 50 nm (buffer used; 20 mM Tris–HCl pH 7.4, 200 mM KCl,5 mM MgCl2). (C) Western blot analyses of a SepF–FtsZ co-elutionexperiment using different MBP–SepF mutants. The columns wereincubated with (þ ) or without (�) B. subtilis FtsZ. FtsZ-antiserumwas used to stain the blots (see Materials and methods, for details).



organization of FtsZ protofilaments by SepF rings is impor-

tant for correct cell division.

Discussion

This is the first report of a protein polymer that directly

supports bundling of FtsZ polymers. Even more surprising is

the fact that this polymer closes into a ring. There are other

cell division proteins that have been shown to promote

bundling of FtsZ filaments, including ZapA of B. subtilis

and E. coli (Gueiros-Filho and Losick, 2002; Small et al,

2007), and the E. coli protein ZipA (RayChaudhuri, 1999;

Hale et al, 2000), but the FtsZ bundles formed by these

proteins do not display the regular, highly organized structure

seen for FtsZ–SepF tubules.

The formation and shape of FtsZ–SepF tubules is reminis-

cent of eukaryotic microtubules, although with a width of

about 25 nm (Desai and Mitchison, 1997), microtubules are

considerably thinner than FtsZ–SepF tubules. The switch

from polymerization to depolymerization in microtubules

is accompanied by a transition from ends with straight

protofilaments to ends with protofilaments curved out-

wards (Mandelkow et al, 1991; Desai and Mitchison, 1997).

A similar phenomenon is observed with FtsZ–SepF tubules.

During the first minutes of the reaction, filaments emanating

from the ends of short tubules tended to be straight, but when

the reaction progressed tubules were seen with splayed tips

with filaments curving outwards (Figure 4). This is likely due

to a shift from GTP to GDP bound FtsZ, as FtsZ protofila-

ments with bound GTP favour a straight conformation,

whereas they tend to become curved when GTP is hydrolyzed

to GDP (Lu et al, 2000). This curvature of FtsZ–SepF tubules

did not result in depolymerization of the tubules, like the

depolymerization phase (dynamic instability) of microtu-

bules, and after prolonged incubation the tubules disintegrate

over their entire length (data not shown). In yeast, the Dam1

kinetochore complex involved in chromosome segregation

forms ring-like structures around microtubules (Wang et al,

2007). Interestingly, the diameter of these rings is about

50 nm, close to that of SepF rings. However, the Dam1

subunits are composed of 10 different proteins. We are

unaware of any other protein that can make such large

regular ring structures by itself.

FtsZ is an abundant protein, yet only a third of the FtsZ

molecules in B. subtilis cells is part of the Z-ring, and it has

been estimated that the Z-ring can be 2–3 protofilaments

thick when division initiates (Anderson et al, 2004).

Considerably more filaments are visible in the FtsZ–SepF

tubules (Figure 3D), so there seems to be insufficient FtsZ

in the cell to make complete tubules when the Z-ring is

formed. Possibly, at a later stage in division, when the

diameter of the Z-ring is much smaller, a more tubular

configuration appears. Although this is speculation, it is

clear that SepF rings support the alignment of FtsZ filaments

which, considering the malformed septa in sepF mutants

(Hamoen et al, 2006), seems to be crucial for proper septum

synthesis.

The striking appearance of SepF rings raises the question

whether there is a biological advantage in employing a

protein that polymerizes into a circular configuration. Why

would the cell not use linear protein polymers to organize the

alignment of FtsZ protofilaments? A possible reason is

depicted in Figure 9. Firstly, it is very difficult to control the

length of a linear polymer (red rods), and when such polymer

becomes exceedingly long the FtsZ polymers (grey rods) will

be spaced too widely (Figure 9A). Polymerization into a ring

prevents this polymer length issue (Figure 9B). However,

even when the linear polymers are somehow of fixed length,

it would still be difficult to control the width of the assembled

super structure, as FtsZ protofilaments alignment would

be able to continue sideways (arrows). This problem is

also circumvented when the supporting polymer adopts a

circular configuration. In Figure 9B SepF is depicted as rings.

Importantly, in this model, even if SepF polymerization is

incomplete, and shorter curved polymers are formed, binding

of these SepF arcs would still result in a tubular alignment of

FtsZ protofilaments.

A

C

E

B

MBP – SepF

FtsZ

wt– + – + – + – + – +

A98V F124S G135NΔ134

+ FtsA – FtsA

wt

G135N�sepF

D

�sepF G135N168

G135N G135N + FtsZ

Figure 8 Characteristics of a SepF mutant defective in ring forma-tion. (A) SepF–FtsZ co-elution experiment using different MBP–SepF mutants. The elution fractions where analyzed by SDS–PAGEand Coomassie staining. The columns were incubated with (þ ) orwithout (�) B. subtilis FtsZ. As a negative control A98V and F124Swere included. (B) Localization of SepF–G135N–GFP fusion inDsepF B. subtilis cells. The fusion protein (white bands) is locatedat cell division sites. (C) Images of negatively stained purifiedSepF–G135N (left panel), and in the presence of FtsZ (rightpanel, FtsZ protofilaments are clearly visible). Scale bar: 200 nm.(D) Mutation G135N is unable to sustain growth in the absence ofFtsA. (E) Western blot analysis of SepF levels in wild-type B. subtiliscells (wt), a sepF knockout strain (DsepF), and a strain expressingSepF–G135N (G135N).



SepF is found only in Gram-positive bacteria and cyano-

bacteria, and not in Gram negatives. One clear difference

between these groups of organisms lies in the thickness of

their cell wall and the way division septa are formed.

Gram-negative bacteria have a thin layer of peptidoglycan,

which follows the constriction of the inner membrane during

division. Gram-positive bacteria have a thick cell wall and

synthesize a continuous thick septum along the division site.

The absence of SepF results in septa which are unusually

thick and often badly malformed (Figure 9C; Supplementary

Figure S6). This seems to be characteristic for sepF mutants,

and is not observed with other cell division mutants, includ-

ing minC, and ezrA (Barak et al, 1998; Hamoen et al, 2006).

We have also checked whether an ftsA deletion causes

irregular septa in B. subtilis, and although the frequency of

septation was clearly reduced, the septa looked normal

(Supplementary Figure S6). We propose that SepF rings

provide a mechanism for arranging FtsZ protofilaments into

higher order structures of defined width, required for the

proper synthesis of the thick division septa in Gram-positive

bacteria and cyanobacteria.

Materials and methods

Bacterial strains, growth conditions, and mediaThe B. subtilis strains and primers used in this study are listed inSupplementary Tables S1 and S2. Bacillus strains were grown at 30and 371C in LB medium, Difco antibiotic medium 3 (PAB), or caseinhydrolysate (CH) medium (Sterlini and Mandelstam, 1969) supple-mented with adenine and guanosine (required for growth ofCRK6000; 20mg/ml each). Bacillus transformants were selectedon PAB plates supplemented with 50mg/ml spectinomycin, 5mg/mlchloramphenicol, 1mg/ml erythromycin, and/or 25mg/ml lincomycin.

Protein purificationB. subtilis sepF was cloned into pMal-c2X (New England Biolabs),using restriction free cloning (van den Ent and Lowe, 2006). The

Xa-cleavage site was positioned immediately upstream of SepF, sothat native SepF, with no additional residues, remains aftercleavage. Overnight cultures of E. coli BL21 (DE3) carryingpMal–SepF were diluted 1:100 in fresh 2� TY medium. Cultureswere grown to an OD600 of 0.4, and MBP–SepF expression wasinduced with IPTG for 3 h. Cell lysates were obtained using a FrenchPress. In total, 1 mM PMSF and 7.5 unit/ml Benzonase (NewEngland Biolabs) were added, and cell debris was removed bycentrifugation. The total protein fraction was loaded onto anamylose column, and MBP–SepF was eluted with elution buffer(20 mM Tris–HCl pH 7.4, 200 mM NaCl, 0.5 mM DTT, and 10 mMmaltose). MBP was cleaved from SepF with Factor Xa (1 mg per250mg fusion protein) overnight at 41C in elution buffer containing2 mM CaCl2. The final purification was performed on an MonoQcolumn followed by a protein concentration step using MilliporeAmicon Ultra protein concentration tubes with a 10-kDa cutoff.Small aliquots were frozen in liquid N2.

B. subtilis FtsZ was purified as described previously (Wang andLutkenhaus, 1993; Scheffers, 2008).

Size exclusion chromatographyPurified SepF (70mg in 200 ml) was loaded onto a Superose 6 10/300column (GE Healthcare), pre-equilibrated with buffer containing20 mM Tris–HCl pH 7.4, 250 mM KCl, and 1 mM EDTA. Sampleswere eluted with 25 ml buffer at a flow rate of 0.3 ml/min. Elutionwas monitored at 280 nm. The experiments were repeated threetimes for each SepF variant, and a representative chromatogram isshown. The Superose 6 column was calibrated with thyroglobulin(669 kDa), aldolase (158 kDa), and RNaseA (13.7 kDa) as molecularstandards (HMV Gel Filtration Calibration Kit, Amersham Pharmacia).

Intracellular concentration of SepF and FtsZThe intracellular concentrations of SepF and FtsZ were determinedusing quantitative western blot analyses as previously described(Ishikawa et al, 2006). We raised rabbit polyclonal antiserumagainst purified SepF. B. subtilis cells were grown in LB medium at371C, and at OD600 of 0.3 a 10-ml sample was taken. Aftercentrifugation, the cells were broken by sonication, mixed with SDSsample buffer, and after heating, loaded onto a SDS–PAA gel forwestern blot analysis. Titrations of purified SepF and FtsZwere loaded onto the same gel (Supplementary Figure S1). Theintensities of the bands were quantified using ImageJ software.Molecules per cell were determined based on CFU of the cultures(2.39Eþ 07/ml), and assuming a cell volume of 2.6 mm3 (Henriqueset al, 1998).

Sedimentation assaysFtsZ sedimentation assays were performed as previously described(Mukherjee and Lutkenhaus, 1998; Scheffers, 2008). Buffers usedwere standard pH 6.5 polymerization buffer (50 mM MES–NaOH pH6.5, 50 mM KCl, 10 mM MgCl2), and polymerization buffer of pH 7.4(50 mM Tris–HCl pH 7.4, 300 mM KCl, 10 mM MgCl2). FtsZ (10 mM)and SepF (6 mM) were incubated for 5 min at room temperature in45ml polymerization buffer. FtsZ polymerization was initiated bythe addition of 1 mM GTP and samples were incubated for 5–20 minat 301C. High molecular weight assemblies were spun down(10 min, 80 000 r.p.m., 251C) in a Beckman ultracentrifuge with aTLA-100 rotor. Supernatant and pellet fractions were separated bySDS–PAA gels followed by colloidal Coomassie staining. Bandintensities were analyzed by ImageJ. The increase in pelleting wasdetermined by subtracting the amount of pelleted protein infractions without GTP from fractions that contained GTP (tostimulate FtsZ polymerization).

Light scattering assaysLight scattering assays were performed as previously described(Mukherjee and Lutkenhaus, 1999; Scheffers, 2008), using a VarianCary Eclipse fluorescence spectrophotometer, and the same reactionconditions as described for the sedimentation assays. In all, 1.5 or2.5 nm excitation and emission slit values were used.

GTP hydrolysis assaysGTPase assays were performed as previously described (Lanzettaet al, 1979; Scheffers, 2008), using Malachite green to detect thereleased phosphate. The reaction conditions were the same as usedfor the sedimentation assays.

wt

�sepF

CBA

Figure 9 A model explaining the benefits of SepF rings in FtsZprotofilament assembly. Protein polymers that bind and align FtsZprotofilaments (grey rods) are depicted in red. (A) Assembly of theFtsZ–SepF structure can expand in all directions (arrows) when thesupporting polymers (red) are linear, whereas (B) protein ringswould force the assembly into one direction. (C) Deletion of sepFresults in deformed division septa (Hamoen et al, 2006).



Electron microscopyFtsZ polymerization was performed as described above, and SepFprotein samples were prepared in polymerization buffers (10mMFtsZ and 6mM SepF). In all, 2 ml samples were applied to glow-discharged 200 mesh carbon coated grids. Grids were negativelystained, using 100 ml uranyl-acetate (2%) and imaged in a PhilipsCM100 electron microscope.

For transmission electron microscopy, cultures were grown in CHmedium at 371C to mid-exponential phase and fixed by the additionof gluteraldehyde to a final concentration of 2%. Cells were thenpelleted and fixation was continued overnight at 41C. Pellets werewashed 3 times with 100 mM phosphate buffer for 15 min, post-fixed with 1% osmium tetroxide in 100 mM phosphate buffer, andincubated for 1 h at 41C. Pellets were then washed in phosphatebuffer, dehydrated in acetone, and embedded in resin that wasallowed to polymerize overnight at 651C. Sections of 80 nm were cutusing an ultracut microtome (Reichert and Jung), negatively stainedwith 2.5% uranyl-acetate and examined with a Philips CM-100electron microscope.

Library constructionFor PCR mutagenesis, the sepF gene containing the Shine–Dalgarnosequence was amplified by PCR with TaKaRa Ex TaqTM (Takara),using SDYlmFF and ylmFgwR (Ishikawa et al, 2006). The reactionwas carried out in the reaction mixture containing 0.1 mM MnCl2(Cromie et al, 1999). Plasmid library was constructed using theGateway cloning technology (Invitrogen). The pX-GW plasmid(K Hiramatsu and S Ishikawa, unpublished) was employed to insertthe mutagenized sepF fragments, under control of the xylose-inducible promoter Pxyl, into the amyE locus of B. subtilis.

Construction of SepF mutantsFor the GFP or MBP-fusions, the A98V, F124S, G135N, and D134mutations were generated using the Quickchange site-directedmutagenesis method (Stratagene). Briefly, plasmid pSepF–GFP(Hamoen et al, 2006) and pMal–SepF were used as templates fortemperature-cycled amplification with primers designed to intro-duce mutations into these codons. Amplification was carried outwith Pfu polymerase (Strategene) and primers (SupplementaryTable S2, position of mutation underlined). The product was treatedwith DpnI to digest unamplified DNA, and transformed intocompetent DH5a cells. The resulting plasmids with the desiredmutations were verified by sequence analysis. To introduce sepF–G135N into B. subtilis, the C-terminal part of sepF was amplified byPCR using pMal–sepFG135N and pMal–sepF (for wild-type control)as template and primers LH224 and LH225 (Supplementary TableS2). The PCR products were digested with EcoRI and BamHI andcloned into the Campbell-integration vector pMutin4 (Vagner et al,1998), creating pM–sepFG135 and pM–sepF.

The SepF mutations were introduced into different B. subtilisbackgrounds by transformation of competent cells as described byMoriya et al (1998). The resulting strains are listed in Supplemen-tary Table S1.

Microscopic imagingFor fluorescence microscopy, overnight cultures were diluted intofresh PAB medium (for GFP–FtsZ) or CH medium (for SepF–GFP),and grown to exponential phase at 301C. Cells were mounted onmicroscope slides covered with a thin film of 1.2% agarose in water.

Images were acquired with a Sony Cool-Snap HQ cooled CCDcamera (Roper Scientific) attached to a Zeiss Axiovert 200Mmicroscope. The images were analyzed with METAMORPH version6 software.

FtsZ co-elutionCell pellets from 30 ml E. coli cultures expressing MBP–SepFmutants were resuspended in 4 ml of buffer A (20 mM Tris–HClpH 7.4, 200 mM KCl, 1 mM EDTA) with 1 mM PMSF. The suspensionwas then sonicated (10 W, 4 s pulses) for a total period of 4 min. Theresulting suspension was centrifuged at 13 000 r.p.m. for 30 min toremove cell debris. Supernatants containing the different MBP–SepFfusion proteins were added to 1.0 ml of amylose resins (BioLabs)pre-equilibrated with buffer A, and the suspensions were gentlymixed at 41C for 30 min. After incubation, the resins were allowedto settle down by gravity flow and washed six times with 2 ml ofbuffer A. After the last wash step the resin was mixed with 0.5 mlbuffer A and one third of the suspension (0.5 ml) was transferred toempty column, to determine the amount of bound MBP–SepF.MBP–SepF was eluted with 2� 0.5 ml elution buffer (buffer Acontaining 10 mM maltose and 1% SDS, 10 min incubation), and theprotein concentration was determined with BCA Protein Assay kit(Thermo Scientific Pierce). For the FtsZ co-elution experiments theresin volume was adjusted so that equal amounts of MBP–SepFwere used. The MBP–SepF containing amylose resin was loaded inempty columns and 4 ml of cell extract from E. coli cells expressingB. subtilis FtsZ was added and incubated for 30 min at 41C. Afterincubation, the columns were washed six times with buffer A beforeelution with 1.5 ml elution buffer. The flow through, first wash step,second wash step, last wash step, and elution fractions wereanalyzed by SDS–PAGE. When necessary, the presence of FtsZ inthe elution fraction was detected with western blotting using FtsZantiserum.

Supplementary dataSupplementary data are available at The EMBO Journal Online(http://www.embojournal.org).

Acknowledgements

We thank the members of CBCB and Shu Ishikawa for helpfuldiscussions. Tracey Davey, Vivian Thompson, Simon Ringgaard andCarolyn Moores are acknowledged for their technical assistance,and help with the interpretation of EM data; Paul Race for his helpwith light scattering and circular dichroism experiments. Thisresearch was supported by a Marie Curie Early Stage ResearchTraining (EST) fellowship (MG, NP), a grant from the BBSRC (JE),a Wellcome Trust Research Career Development Fellowship (LWH),a KAKENHI grant-in-aid for scientific research in the PriorityArea ‘Systems Genomics’ from the Ministry of Education, Culture,Sports, Science, and Technology of Japan (NO), and a grant (PTDC/BIA-MIC/098637/2008) from the Fundacao para a Ciencia eTecnologia (DJS).

Conflict of interest

The authors declare that they have no conflict of interest.

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large ring polymers align ftsz polymers for normal septum formation

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